D-2-Hydroxy-4-Methylvalerate Dehydrogenase from Lactobacillus Delbrueckii Subsp. Bulgaricus- I. Kinetic Mechanism and pH Dependence of Kinetic Parameters, Coenzyme Binding and Substrate Inhibition (original) (raw)
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European Journal of Biochemistry, 1994
A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD+-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD+-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage P, promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched P-carbon. NAD+-dependent 2-hydroxyacid dehydrogenases of broad substrate specificity have been reported in many species [l]. They catalyse the stereospecific and reversible reduction of various aliphatic and aromatic 2-oxocarboxylic acids to the corresponding 2-hydroxycarboxylic acids. The optically active products are not only valuable compounds as synthons for industrial processes as, for instance, in the production of semisynthetic antibiotics or pharmaceuticals [2], but they can also be used for clinical analysis of blood samples [3]. These enzymes can be divided into two groups on the basis of their substrate preference: mandelate dehydrogenase (or benzoylformate reductase), on the one hand, and 2-hydroxyisocaproate (i.e. 2-hydroxy-4-methylpentanoate) dehydrogenase (or 2-oxoisocaproate reductase), on the other hand. Each class can be further subdivided according to the D-or L-stereoisomer produced. Their actual substrate and function in vivo are not known.
NAD+‐Dependent d‐2‐Hydroxyisocaproate Dehydrogenase of Lactobacillus Delbrueckii subsp. Bulgaricus
European Journal of Biochemistry, 1994
A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD+‐dependent d‐2‐hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333‐residue protein closely related to D‐2‐hydroxyisocaproate (i.e. 2‐hydroxy‐4‐methyl‐pentanoate) dehydrogenase (d‐HO‐HxoDH) of Lactobacillus casei and other NAD+‐dependent d‐lactate dehydrogenases (d‐LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two‐step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2‐oxoacid substrates exce...
An Investigation of the Active Site of Lactate Dehydrogenase with NAD+ Analogues
European Journal of Biochemistry, 1981
The kinetic properties of 18 NAD' analogues, with alterations to the nicotinamide moiety, have been studied with respect to dogfish M4, rabbit M4 and beef H4 lactate dehydrogenases. The size of the groups present at C-3 of the pyridinium can be increased quite extensively without loss of coenzyme activity. Groups tested were thioamide, methyl, ethyl, diazoketone and chloroacetyl. Substitutions at positions C-4 and C-5 prevent proper positioning for hydride transfer and can hinder binding to the enzyme. The kinetic properties of pyridine-adenine dinucleotide and its 3-iodo derivative reveal the binding role of the amide at C-3 whereas 3-cyanopyridine-adenine dinculeotide is a strong inhibitor.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008
A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na + /H + antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na + -resistance on an antiporterdeficient E. coli strain, nor did they confer Na + /H + antiport activity in vesicle assays. The purified hexahistidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B. . DNA sequences: new sequence for Bp ndh-2A and ndh-2B were deposited Genbank accession no. EU030627 and EU030628
A general method for relieving substrate inhibition in lactate dehydrogenases
Protein Engineering Design and Selection, 1999
The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate. Since this is the third enzyme to show this behaviour, we suggest that the S163L mutation is a general method for the removal of substrate inhibition in L-LDH enzymes. Engineering such enzymatic properties has clear industrial applications in the use of these enzymes to produce enantiomerically pure α-hydroxy acids. The mutation leads to two principal effects. (1) Substrate inhibition is caused by the formation of a covalent adduct between pyruvate and the oxidized form of the cofactor. The inability of S163L mutants to catalyse the formation of this inhibitory adduct is demonstrated. However, NMR experiments show that the orientation of the nicotinamide ring in the mutant NAD ⍣ binary complex is not perturbed. (2) The mutation also leads to a large increase in the K M for pyruvate. The kinetic and binding properties of S163L LDH mutants are accounted for by a mechanism which invokes a non-productive, bound form of the cofactor. Molecular modelling suggests a structure for this non-productive enzyme-NADH complex.
Purification and properties of NADH dehydrogenase from an alkalophilic bacillus
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1983
Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75°C. A 2,100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95,000, consisting of two 50,000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2,6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.
Journal of Molecular Biology, 2008
2-(Hydroxymethyl)glutarate dehydrogenase, the fourth enzyme of the anaerobic nicotinate fermentation pathway of Eubacterium barkeri, catalyzes the NADH-dependent conversion between (S)-2-formylglutarate and (S)-2-(hydroxymethyl)glutarate. As shown by its 2.3-Å crystal structure, this enzyme is a novel member of the β-hydroxyacid dehydrogenase family and adopts a tetrameric architecture with monomers interacting via their C-terminal catalytic domains. The NAD-binding domains protrude heterogeneously from the central, tetrameric core with domain rotation angles differing up to 12°. Kinetic properties of the enzyme, including NADH inhibition constants, were determined. A strong NADH binding in contrast to weaker NAD + binding of the protein was inferred from fluorometrically determined binding constants for the dinucleotide cofactor. The data support either an Iso Ordered Bi Bi mechanism or a more common Ordered Bi Bi mechanism as found in other dehydrogenases.
Journal of Molecular Biology, 1997
D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei is a homodimer with 333 amino acids and a molecular mass of 37 kDa per subunit. The enzyme belongs to the protein family of NAD-dependent D-2-hydroxycarboxylate dehydrogenases and within this family to the subgroup of D-lactate dehydrogenases (D-LDHs). Compared with other D-LDHs D-HicDH is characterized by a very low speci®city regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates. Hexagonal crystals of recombinant D-HicDH in the presence of NAD and 2oxoisocaproate (4-methyl-2-oxopentanoate) were grown with ammonium sulphate as precipitating agent. The structure of these crystals was solved by molecular replacement and re®ned to a ®nal R-factor of 19.6% for all measured X-ray re¯ections in the resolution range (I to 1.86 A Ê). Both NAD and 2-oxoisocaproate were identi®ed in the electron density map; binding of the latter in the active site, however, competes with a sulphate ion, which is also de®ned by electron density. Additionally the ®nal model contains 182 water molecules and a second sulphate ion. The binding of both an in vitro substrate and the natural cosubstrate in the active site provides substantial insight into the catalytic mechanism and allows us to assess previously published active site models for this enzyme family, in particular the two most controversial points, the role of the conserved Arg234 and substrate binding. Furthermore the overall topology and details of the D-HicDH structure are described, discussed against the background of homologous structures and compared with one closely and one distantly related protein.